The present invention relates to xcex1-2,8-sialyltransferase, a cDNA coding for the xcex1-2,8-sialyltransferase, a recombinant vector containing the cDNA as an insert and a cell harboring the recombinant vector as well as methods of producing same. The invention further relates to a method of producing carbohydrate chains using the xcex1-2,8-sialyltransferase and to a method of producing carbohydrate chains through production of the xcex1-2,8-sialyltransferase in transformant cells. Still further, it relates to a method of detecting the xcex1-2,8-sialyltransferase and a method of inhibiting the production of the xcex1-2,8-sialyltransferase, each using DNA coding for the xcex1-2,8-sialyltransferase of the invention. The xcex1-2,8-sialyltransferase of the invention is useful, in particular, in the production of carbohydrate chains having a useful physiological activity, for example the ganglioside GD3, and modifications thereof.
While proteins produced in prokaryotes, for example Escherichia coli, have no carbohydrate chain, proteins and lipids produced in eukaryotes such as yeast, fungi, plant cells and animal cells have a carbohydrate chain bound thereto in many instances.
Carbohydrate chains bound to proteins in animal cells include N-glycoside bond type carbohydrate chains (also called N-glycans) bound to an asparagine (Asn) residue in the protein and O-glycoside bond type carbohydrate chains (also called O-glycans) bound to a serine (Ser) or threonine (Thr) residue. It has recently been revealed that a certain kind of lipid containing a carbohydrate chain is covalently bound to a number of proteins and that those proteins are attached to the cell membrane through the lipid. This carbohydrate chain-containing lipid is called glycosyl phosphatidylinositol anchor.
Other carbohydrate chains, including glycosaminoglycans, are also present in animal cells. Compounds comprising a protein covalently bound to a glycosaminoglycan are called proteoglycans. The glycosaminoglycans of the carbohydrate chains of proteoglycans are similar in structure to O-glycans, which are carbohydrate chains of glycoproteins, but differ chemically therefrom. Glycosaminoglycans comprise repeating disaccharide units composed of glucosamine or galactosamine and a uronic acid (except for keratan sulfate which has no uronic acid residue) and have a covalently bound sulfate residue (except for hyaluronic acid which has no sulfate residue).
Further, carbohydrate chains in animal cells are also present in substances called glycolipids. Sphingoglycolipids are one type of glycolipid present in animal cells. Sphingoglycolipids are composed of a carbohydrate, a long-chain fatty acid and sphingosine, a long-chain base, covalently bound together. Glyceroglycolipids are composed of a carbohydrate chain and glycerol covalently bound together.
Recent advances in molecular biology and cellular biology have made it possible to clarify the functions of carbohydrate chains. To date, a variety of functions of carbohydrate chains have been elucidated. First, carbohydrate chains play an important role in the clearance of glycoproteins in blood. It is known that erythropoietin produced by introducing the relevant gene into Escherichia coli retains activity in vitro but undergoes rapid clearance in vivo [Dordal et al.: Endocrinology, 116, 2293 (1985) and Browne et al.: Cold Spring Harbor Symposia on Quantitative Biology, 51, 693 (1986)]. It is known that while native human granulocyte-macrophage colony stimulating factor (hGM-CSF) has two carbohydrate chains of the N-glycoside bond type, a reduction in the number of carbohydrate chains results in a proportional increase in the rate of clearance in rat plasma [Donahue et al.: Cold Spring Harbor Symposia on Quantitative Biology, 51, 685 (1986)]. The rate of clearance and the site of clearance may vary or differ depending on the structure of the carbohydrate chain in question. For example, it is known that hGM-CSF having a sialic acid residue undergoes clearance in the kidney while hGM-CSF deprived of sialic acid shows an increased rate of clearance and undergoes clearance in the liver. Alpha1-acid glycoproteins differing in carbohydrate structure and biosynthesized in the presence of various N-glycoside type carbohydrate chain biosynthesis inhibitors using a rat liver primary culture system were studied with respect to their rate of clearance from rat plasma and their rate of clearance from rat perfusate. In both cases, the rate of clearance was reduced in the order: high mannose type, carbohydrate chain-deficient type, hybrid type and composite type (natural type). It is known that the clearance from blood of tissue-type plasminogen activator (t-PA), which is used medicinally as a thrombolytic agent, is greatly influenced by the structure of its carbohydrate chain.
It is known that carbohydrate chains give protease resistance to proteins. For example, when carbohydrate formation on fibronectin is inhibited with tunicamycin, the rate of degradation of intracellular carbohydrate chain-deficient fibronectin increases. It is also known that addition of a carbohydrate chain may result in increased heat stability or freezing resistance. In the case of erythropoietin and xcex2-interferon, among others, the carbohydrate chain is known to contribute to increased solubility of the protein.
Carbohydrate chains also serve to maintain protein tertiary structure. It is known that when the membrane binding protein of vesicular stomatitis virus is devoid of the two naturally-occurring N-glycoside bond type carbohydrate chains, transport of the protein to the cell surface is inhibited and that when new carbohydrate chains are added to the protein, it is transported. It was revealed that, in that case, intermolecular association of the protein through disulfide bonding is induced following the elimination of carbohydrate chains and, as a result, protein transport is inhibited. When carbohydrate chains are added, association is inhibited, and the proper tertiary protein structure is maintained and protein transport becomes possible. As regards the site of addition of the new carbohydrate, it has been shown that there is a considerable amount of flexibility. In contrast, it has also been shown in certain instances that, depending on the site of carbohydrate chain introduction, the transport of a protein having a natural carbohydrate chain or chains may be completely inhibited.
Examples are also known where a carbohydrate chain serves to mask an antigenic site of a polypeptide. In the case of hGM-CSF, prolactin, interferon-xcex3, Rauscher leukemia virus gp70 and influenza hemagglutinin, experiments using a polyclonal antibody or a monoclonal antibody to a specific site on the peptide suggest that carbohydrate chains of these proteins inhibit antibody binding. Cases are also known where carbohydrate chains themselves are directly involved in the expression of activity by aglycoprotein. For instance, carbohydrates are thought to be associated with the expression of activity of such glycoprotein hormones as luteinizing hormone, follicle stimulating hormone and chorionic gonadotropin.
Carbohydrate chains serve an important function in the phenomenon of recognition between cells, between proteins or between a cell and a protein. For example, it is known that structurally different carbohydrate chains undergo clearance in vivo at different sites. It has recently been revealed that the ligand of the protein ELAM-1, which is expressed specifically on vascular endothelial cells during an inflammatory response and promotes adhesion to neutrophils, is a carbohydrate chain called sialyl Lewis x [NeuAcxcex12-3Galxcex21-4(Fucxcex11-3)GlcNAc; where NeuAc: sialic acid; Gal: galactose; Fuc: fucose; GlcNAc: N-acetylglucosamine]. The possible use of carbohydrate chains themselves or modifications thereof as drugs or the like is thus suggested [Phillips et al.: Science, 250, 1130 (1990); Goelz et al.: Trends in Glycoscience and Glycotechnology, 4, 14 (1992)]. Like ELAM-1, L-selectin, expressed in some T lymphocytes or neutrophils, and GMP-140 (also called P-selectin), expressed in platelets or on the membrane surface of vascular endothelial cells upon inflammatory stimulation, are associated with inflammatory responses. It is suggested that their ligand may be a carbohydrate chain analogous to sialyl Lewis x, the ELAM-1 ligand [Rosen et al.: Trends in Glycoscience and Glycotechnology, 4, 1 (1992); Larsen et al.: Trends in Glycoscience and Glycotechnology, 4, 25 (1992); Aruffo et al.: Trends in Glycoscience and Glycotechnology, 4, 146 (1992)].
It has been suggested that, in cancer metastatis, as in inflammatory responses, ELAM-1 and GMP-140 cause adhesion of cancer cells to the vascular endothelium or aggregation of cancer cells with platelets and thereby promote cancer metastatis [Goelz et al.: Trends in Glycoscience and Glycotechnology, 4, 14 (1992); Larsen et al.: Trends in Glycoscience and Glycotechnology, 4, 25 (1992)]. This is in agreement with the finding that the level of expression of the sialyl Lewis x carbohydrate chain is high in cancer cells that are highly metastatic [Irimura et al.: Jikken Igaku (Experimental Medicine), 6, 33 (1988)].
Gangliosides constitute a group of cell membrane constituent glycolipids. They are molecules composed of a sialic acid residue-containing carbohydrate chain, which is a hydrophilic side chain, sphingosine, which is a hydrophobic side chain, and a fatty acid. It is known not only that the expression of gangliosides varies with the cell, organ and animal species but also that gangliosides undergo quantitative and qualitative changes during the process of cell differentiation or oncogenesis [Hakomori: Cancer Research, 45, 2405 (1985)]. Scores of gangliosides have been discovered so far, including GM3 which is expressed in a variety of normal cells, and gangliosides occurring in extremely small amounts [Wiegant: Gangliosides and Cancer, Verlagsgesellschaft, 1989, pages 5-15]. GD3, for example, occurs in small amounts in normal tissues but it is expressed at high levels in neuroectodermal tumors, such as malignant melanoma. It is therefore believed to be a type of cancer antigen [Tsuchida et al.: Journal of the National Cancer Institute, 78, 45-54 (1987)]. A recent report shows that the proportions of GD3 and GM3 vary according to the degree of malignancy of malignant melanoma [Ravindranath et al.: Cancer, 67, 3029 (1991)] and it is widely known that GD3 is an important cancer antigen. Furthermore, it has been demonstrated that the expression of GD3 is induced in cells into which an oncogene has been introduced, supporting the close relation between cell transformation and GD3 expression [Sanai et al.: Journal of Biochemistry, 107, 740-742 (1990]. As for the functions of GD3, it has been suggested that it plays an important role in adhesion of cancer cells to extracellular substrates [Burns et al.: Journal of Cell Biology, 107, 1225-1230 (1988)].
It has been suggested that abnormal expression of GD3 is due to xcex1-2,8-sialyltransferase, which is a GD3 synthetase [Yusuf et al.: Biological Chemistry Hoppe-Seyler, 368, 455-462 (1987)]. Only the partial purification of GD3 synthetase has been reported [Gu et al.: Biochemical and Biophysical Research Communications, 166, 387-393 (1990)]. No GD3 synthetase has been isolated as yet.
Attempts have been made to effect passive immunization of cancer patients by administering a monoclonal antibody to GD3 [Houghton et al.: Proceedings of the National Academy of Sciences of the U.S.A., 82, 1242 (1985)] and to effect active immunization of cancer patients by administering GD3 per se as a vaccine [Portoukalian et al.: International Journal of Cancer, 49, 893-899 (1991); Ritter et al.: International Journal of Cancer, 48, 379-385 (1991)]. GD3 is thus a valuable cancer antigen. The quantity of GD3 that can be obtained by purification from tissues, however, is limited [Takamizawa et al.: Journal of Biological Chemistry, 261, 5625-5630 (1986)]. Chemical synthesis of GD3 requires sophisticated techniques and yields are very low [Ito et al.: Journal of the American Chemical Society, 111, 8508-8510 (1989)].
In view of the above-described association of GD3 with oncogenesis or cancer metastasis, it is expected that cancer might be treated by inhibiting the enzymatic activity of the GD3 synthetase xcex1-2,8-sialyltransferase or suppressing expression of the relevant gene. Antisense RNA/antisense DNA techniques [Tokuhisa: Bioscience and Industry, 50, 322 (1992); Murakami; Kagaku (Chemistry), 46, 681 (1991)] and triple helix techniques [Chubb and Hogan: Trends in Biotechnology, 10, 132 (1992) can be used to suppress gene expression specifically. For suppressing expression of a specific glycosyltransferase using the antisense RNA/DNA technique, the gene in question or information about the base sequence of the gene is required. It is thus important to clone the desired glycosyltransferase gene and determine the base sequence of same.
Further, as mentioned above, GD3 synthetase xcex1-2,8-sialyltransferase is associated with oncogenesis and it is thus expected that it could be used in cancer diagnosis, that is, that the level of expression of the synthetase could be used to detect the presence of a tumor. The following can be used to assay expression of the xcex1-2,8-sialyltransferase (GD3 synthetase) gene: Northern hybridization using the gene in a labeled form, for example in a radiolabeled form, as a probe [Sambrook, Fritsch and Maniatis: Molecular Cloningxe2x80x94A laboratory manual, second edition, Cold Spring Harbor Laboratory Press, 1989] and polymerase chain reaction (hereinafter, xe2x80x9cPCRxe2x80x9d) [Innis et al.: PCR Protocols, Academic Press, 1990]. In applying these techniques, the gene for the GD3 synthetase xcex1-2,8-sialyltransferase or knowledge of the base sequence thereof is required. From this viewpoint as well, it is important to clone the gene for GD3 synthetase xcex1-2,8-sialyltransferase and determine its base sequence.
JP-A-2-227075 discloses the possibility of improving the properties of physiologically active useful proteins, such as granulocyte colony stimulating factor (G-CSF) and prourokinase (pro-UK), by artificially introducing a carbohydrate chain into the proteins using recombinant DNA technology.
It is an important problem from an industrial viewpoint to modify the structure of the carbohydrate chain of a glycoprotein or a glycolipid, or to prepare a specific carbohydrate chain or a modification thereof in large quantities, making use of xcex1-2,8-sialyltransferase activity of the GD3 synthetase.
There have been marked advances in recent years in the means for modifying carbohydrate chain structures. In particular, it is now possible to structurally modify carbohydrate chains using highly specific enzymes (exoglucosidases) that are capable of releasing carbohydrate units one by one from the end of the carbohydrate chain, or glycopeptidases or endoglycosidases that are capable of cleaving the site of binding to the peptide chain without causing any change in either the peptide or carbohydrate chains, and accordingly, to study biological roles of carbohydrate chains in detail. The recent discovery of endoglycoceramidases that are capable of cleaving the glycolipids at the site between the carbohydrate chain and the ceramide [Ito and Yamagata: Journal of Biological Chemistry, 261, 14278 (1986)] has not only made it easy to prepare carbohydrate chains of glycolipids but has also promoted investigations into functions of glycolipids, in particular glycolipids occurring in cell surface layers. Further, it has become possible to add new carbohydrate chains using glycosyltransferases. Thus, for instance, sialic acid can be added to a carbohydrate chain terminus using sialyltransferase (Sabesan and Paulson: Journal of the American Chemical Society, 108, 2068 (1986)]. It is also possible, using various glycosyltransferases or glycosidase inhibitors, to modify carbohydrate chains that are to be added [Allan et al. Annual Review of Biochemistry, 56, 497 (1987)]. However, there is no means available for producing glycosyltransferases for use in synthesizing carbohydrate chains. It is desirable to produce glycosyltransferases in large quantities by cloning glycosyltransferases and causing efficient expression of glycosyltransferases in host cells utilizing recombinant DNA technology.
As far as sialyltransferase is concerned, a gene for an enzyme having xcex2-galactoside xcex1-2,6-sialyltransferase activity has been isolated and the base sequence thereof has been reported [Weinstein et al.: Journal of Biological Chemistry, 262, 17735 (1987)]. As regards an enzyme having xcex2-galactoside xcex1-2,3-sialyltransferase activity, cloning of a gene coding for an enzyme catalyzing the addition of sialic acid to galactose in an O-glycoside bond type carbohydrate chain (carbohydrate chain added to a serine or threonine residue) of glycoproteins has been reported by Gillespie et al. but the base sequence of said gene has not been reported [Gillespie et al.: Glycoconjugate Journal, 7, 469 (1990)]. Weinstein et al. reported a method of purifying an enzyme having xcex2-galactoside xcex1-2,3-sialyltransferase activity from rat liver [Weinstein et al.: Journal of Biological Chemistry, 257, 13835 (1982)]. This method, however, provides the desired enzyme only in very small amounts. This rat liver xcex2-galactoside xcex1-2,3-sialyltransferase gene has been cloned by Wen et al. [Wen et al.: Journal of Biological Chemistry, 267, 21011 (1992)]. There has been no report, however, of the cloning of a gene for human galactoside xcex1-2,8-sialyltransferase. Large scale preparation of a sialyltransferase species having xcex1-2,8-sialyltransferase activity or cloning of a gene for encoding a product having sialyltransferase activity has not been reported as yet. Therefore, no means is currently available for large scale preparation of a sialyltransferase having xcex1-2,8-sialyltransferase activity, in particular human galactoside xcex1-2,8-sialyltransferase. Methods of detecting or suppressing expression of the enzyme have also not been established.
It is an object of the present invention to provide a novel xcex1-2,8-sialyltransferase species that would make possible efficient production of the ganglioside GD3, a cDNA coding for xcex1-2,8-sialyltransferase, and a vector containing that cDNA. Another object is to provide a method of detecting xcex1-2,8-sialyltransferase activity, which method would be useful in the diagnosis or treatment of diseases such as cancer, and a method of suppressing the expression of xcex1-2,8-sialyltransferase.
The present inventors extracted mRNA from the human melanoma cell line WM266-4, synthesized cDNA using the mRNA as a template, inserted the cDNA into an expression cloning vector, introduced the thus-constructed cDNA library into cells, selected, from among the cells obtained, cells strongly reactive with an antibody specific for the ganglioside GD3 using a fluorescence activated cell sorter (hereinafter, xe2x80x9cFACSxe2x80x9d) and thus successfully cloned a gene coding for a novel species of xcex1-2,8-sialyltransferase. They further introduced the xcex1-2,8-sialyltransferase-encoding gene into Namalwa cells to effect expression of the gene and found that the novel xcex1-2,8-sialyltransferase was expressed in the cells and further that the amount of the ganglioside GD3 present on the cell surface increased.
The present invention is described in detail as follows.
The present invention relates, in one embodiment, to a novel xcex1-2,8-sialyltransferase species comprising the amino acid sequence defined in SEQ ID NO:2. The invention further relates to a cDNA coding for xcex1-2,8-sialyltransferase and to a recombinant vector harboring the cDNA, and to a cell containing the recombinant vector. The xcex1-2,8-sialyl-transferase of the present invention catalyzes the addition of a sialic acid residue, in xcex12xe2x86x928 linkage, to a sialic acid residue contained in the ganglioside GM3 which is an acceptor.
DNA sequences coding for the xcex1-2,8-sialyltransferase of the present invention include, (a) DNA comprising the base sequence defined in SEQ ID NO:1; (b) DNA containing a base sequence different from the base sequence defined in SEQ ID NO:1, the difference being due to the availability of a plurality of codons for one amino acid or to spontaneous mutation occurring in individual animals including human; and (c) DNA derived from the DNA defined in (a) or (b) by mutation, such as substitution, deletion or insertion mutation, that does not cause loss of xcex1-2,8-sialyltransferase activity, for example, DNA encoding a modified amino acid sequence derived from the sequence defined in SEQ ID NO:2 from which the first to 56th amino acid residues from the N-terminus are deleted as shown in Example 3 below, or DNA homologous to the xcex1-2,8-sialyltransferase-encoding DNA defined in (a) or (b) that can be isolated by the hybridization. Homologous DNA means DNA obtainable by the colony hybridization or plaque hybridization technique using a DNA containing the base sequence defined in SEQ ID NO:1 as a probe. Specifically, homologous DNA means DNA identifiable by performing hybridization at 65xc2x0 C. in the presence of 0.7-1.0 M sodium chloride using a filter with a colony- or plaque-derived DNA fixed thereon and then washing the filter in a 0.1-fold to 2-fold concentrated SSC solution (1-fold concentrated SSC solution comprising 150 mM sodium chloride and 15 mM sodium citrate) at 65xc2x0 C. The hybridization procedure is described in Molecular Cloningxe2x80x94A Laboratory Manual, 2nd Edition, edited by Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. Any xcex1-2,8-sialyltransferase species encoded by DNAs defined above in (a), (b) and (c) is included in the xcex1-2,8-sialyltransferase of the present invention.
The following describes a method of producing DNA coding for the xcex1-2,8-sialyltransferase of the present invention is described, taking DNA defined above in (a) as an example.
A cDNA library is constructed by inserting cDNA synthesized using mRNA extracted from the animal cell as a template into an expression cloning vector. This cDNA library is introduced into animal cells or insect cells, then cells that react strongly with an antibody specific for the ganglioside GD3 are enriched and isolated utilizing a FACS. The desired xcex1-2,8-sialyltransferase-encoding cDNA is isolated from the cells.
Animal cells suitable for use in the above process can be any cells provided that they are animal cells in which the xcex1-2,8-sialyltransferase of the present invention is expressed. Thus, for instance, the human melanoma cell line WM266-4 (ATCC CRL 1676) can be used. The vector into which the cDNA synthesized using the mRNA extracted from these cells as a template is to be inserted can be any vector provided that it allows insertion thereinto and expression of the cDNA. For instance, pAMoPRC3Sc or the like can be used. The animal or insect cells into which the cDNA library constructed using the vector is introduced can be any cells provided that they allow introduction therein and expression of the cDNA library. Thus, for instance, human Namalwa cells [Hosoi et al.: Cytotechnology, 1, 151 (1988)] or the like can be used. In particular, a direct expression cloning system using Namalwa cells as the host is advantageous in that the efficiency of introduction of a cDNA library into host Namalwa cells is very high and in that the plasmids (cDNA library) introduced can be maintained extrachromosomally in the system and can be readily recovered from the cells obtained by screening using carbohydrate chain-specific antibody and a FACS. Therefore, this system is preferred. The anti-ganglioside GD3 antibody to be used in the practice of the invention can be any antibody provided that it reacts with the ganglioside GD3. For instance, KM-643 (EP-A-0493686) can be used. The animal cells, after introduction thereinto of the cDNA library, are fluorescence-labeled using the anti-GD3 antibody and then cells showing increased binding to the antibody are separated and enriched using a FACS. From the cells thus obtained, a plasmid or DNA fragment containing the cDNA coding for the xcex1-2,8-sialyltransferase of the present invention is recovered using, for example, known methods, e.g. the Hart method [Robert F. Margolskee et al.: Molecular and Cellular Biology, 8, 2837 (1988)]. Plasmids of the invention containing cDNA coding for the enzyme include pUC119-WP1R. Escherichia coli JM105/pUC119-WP1R, an Escherichia coli strain harboring pUC119-WP1R, was deposited, on Feb. 18, 1993, at the National Institute for Bioscience and Human Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305, JAPAN under the deposit number FERM BP-4192 (Budapest Treaty deposit).
The DNA defined above in (b) or (c) can be produced using the well-known recombinant DNA techniques [JP-A-2-227075; Molecular Cloningxe2x80x94A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989; etc.], such as hybridization techniques or methods of introducing mutation into DNA, based on the xcex1-2,8-sialyltransferase-encoding cDNA obtained by the method described above. The xcex1-2,8-sialyltransferase-encoding cDNA of the present invention can also be produced by chemical synthesis.
The xcex1-2,8-sialyltransferase of the present invention can be produced by constructing a recombinant vector by inserting DNA coding for the xcex1-2,8-sialyltransferase of the present invention obtained, for example, by the method described above into an appropriate vector and in operable linkage with a suitable promoter, introducing the recombinant vector into host cells and cultivating the cells obtained. The host cells to be used here can be any host cells suitable ror use in recombinant DNA technology, for example, prokaryotic cells, animal cells, yeasts, fungi and insect cells. An example of a suitable prokaryotic cell is Escherichia coli. CHO cells (Chinese hamster cells), COS cells (sminian cells) and Namalwa cells (human cells) are examples of suitable animal cells.
Vectors into which DNA coding for the xcex1-2,8-sialyltransferase of the present invention is inserted can be any vector provided that it allows insertion therein of the xcex1-2,8-sialyltransferase-encoding DNA and expression of the DNA in host cells. pAGE107 [JP-A-3-22979; Miyaji et al.: Cytotechnology, 3, 133 (1990)], pAS3-3 (JP-A-2-227075), pAMoERC3Sc, and CDM8 [Brian Seed et al.: Nature, 329, 840 (1987)] are examples. For the expression of the enzyme of the present invention in Escherichia coli, a plasmid is preferably used. The foreign DNA is inserted into the plasmid so that it is operably linked to a promoter with potent transcription activity, for example, the trp promoter, and so that the distance between the Shine-Dalgarno sequence (hereinafter, xe2x80x9cSD sequencexe2x80x9d) and the initiation codon is of an appropriate length (for example 6-18 bases). Plasmid pKYP10 (JP-A-58-110600), pLSA1 [Miyaji et al.: Agricultural and Biological Chemistry, 53, 277 (1989)] and pGEL1 [Sekine et al.: Proceedings of the National Academy of Sciences of the U.S.A., 82, 4306)] are specific examples.
Recombinant DNA techniques to be used in the practice of the invention include those described in JP-A-2-227075 or those described in Sambrook, Fritsch, Maniatis et al.: Molecular Cloningxe2x80x94A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989. A number of commercially available kits can be used for mRNA isolation and cDNA library construction. Known methods can be used to introduce the DNA into animal cells. The electroporation method [Miyaji et al.: Cytotechnology, 3, 133 (1990)], the calcium phosphate method (JP-A-2-227075) and the lipofection method [Philip L. Felgner et al.: Proceedings of the National Academy of Sciences of the U.S.A., 84, 7413 (1987)] are examples. Transformant isolation and cultivation can be performed essentially according to the method described in JP-A-2-227075 or JP-A-2-257891.
Suitable methods of producing the xcex1-2,8-sialyltransferase include the method of intracellular production in a host, the method of extracellular production or the method of production on a host cell membrane external layer. The site of production varies depending on the kind of host cell used and the form of the glycosyltransferase to be produced. In cases where animal cells are used as the host and a glycosyltransferase is produced in its native form, the enzyme is generally produced within the host cells or on the host cell membrane external layer and a portion of the enzyme produced is cleaved with protease and secreted extracellularly. The DNA recombination technique of Paulson et al. [C. Paulson et al.: The Journal of Biological Chemistry, 264, 17619 (1989)] and Low et al. [John B. Lowe et al., Proceedings of the National Academy of Sciences of the U.S.A., 86, 8227 (1989); John B. Lowe et al.: Genes and Development, 4, 1288 (1990)] can be used to cause production of the enzyme in a form composed of a glycosyltransferase portion containing the active site and a signal peptide added thereto.
Production of the enzyme can be increased by utilizing a gene amplification system using the dihydrofolate reductase gene, for example, as described in JP-A-2-227075.
Alpha-2,8-sialyltransferase produced in accordance with the present invention can be purified using ordinary methods of purifying glycosyltransferases [J. Evan. Sadler et al.: Methods of Enzymology, 83, 458]. When produced in Escherichia coli, the enzyme can be efficiently purified by a combination of the above method and the method described in JP-A-63-267292. It is also possible to produce the enzyme of the present invention in the form of a fusion protein and to purify the same by affinity chromatography using a substance having affinity for the fused protein. For example, the enzyme of the present invention can be produced fused with protein A. Such a protein can be purified by affinity chromatography using immunoglobulin G, essentially according to the method of Lowe et al. [John B. Lowe et al.: Proceedings of the National Academy of Sciences of the U.S.A., 86, 8227 (1989); John B. Lowe et al.: Genes and Development, 4, 1288 (1990). It is also possible to purify the enzyme by affinity chromatography using an antibody to the enzyme.
The sialyltransferase activity can be determined according to known methods [J. Evan. Sadler et al.: Methods in Enzymology, 83, 458; Bo E. Samuelson: Methods in Enzymology, 138, 567; Manju Basu et al.: Methods in Enzymology, 138, 575; and Nacyuki Taniguti et al.: Methods in Enzymology, 179, 397].
Carbohydrate chains can be synthesized in vitro using the xcex1-2,8-sialyltransferase of the present invention. For example, the nonreducing end of oligosaccharides NeuAc xcex12-3Gal xcex21-4Glc can be provided with a sialic acid residue in xcex12xe2x86x928 linkage. Further, the ganglioside GM3 which serves as a substrate, when treated with the xcex1-2,8-sialyltransferase of the present invention, can be modified to the ganglioside GD3.
By using DNA coding for the xcex1-2,8-sialyltransferase of the present invention and causing simultaneous production of xcex1-2,8-sialyltransferase and a carbohydrate chain (a glycoprotein, glycolipid or oligosaccharide) to serve as an acceptor substrate of the xcex1-2,8-sialyltransferase in animal or insect cells that are producing the carbohydrate chain, it is possible to cause the xcex1-2,8-sialyltransferase produced to act on the carbohydrate chain in the cells to provide a sialic acid residue with a nonreducing end of the carbohydrate chain. For instance, the ganglioside GD3 can be produced by effecting simultaneous production of the xcex1-2,8-sialyltransferase in cells that are producing the ganglioside GM3.
Furthermore, it is also possible to excise, by known enzymatic or chemical techniques, a part of the oligosaccharide from the glycoprotein, glycolipid or oligosaccharide having a modified carbohydrate chain structure as produced in the above manner.
DNA coding for the xcex1-2,8-sialyltransferase of the present invention can be used not only to effect modification of a carbohydrate chain of a protein or glycolipid or to effect efficient production of a specific carbohydrate chain, but also to treat diseases, such as inflammation and cancer metastasis utilizing, for example, antisense RNA/DNA techniques. Such DNA can also be used in the diagnosis of such diseases, for example, utilizing Northern hybridization and PCR techniques.
For instance, DNA coding for the xcex1-2,8-sialyltransferase of the present invention can be used to prevent expression of the xcex1-2,8-sialyltransferase by antisense RNA/DNA technology [Tokuhisa: Bioscience and Industry, 50, 322-326 (1992); Murakami: Kagaku (Chemistry), 46, 681-684 (1991); Miller: Biotechnology, 9, 358-362 (1992); Cohen: Trends in Biotechnology, 10, 87-91 (1992); Agrawal: Trends in Biotechnology, 10, 152-158 (1992)] or triple helix techniques [Chubb and Hogan: Trends in Biotechnology, 10, 132-136 (1992)]. More specifically, based on a part of the base sequence of the DNA coding for the xcex1-2,8-sialyltransferase of the present invention, preferably a base sequence of 10-50 bases in length as occurring in the translation initiation region, an oligonucleotide can be designed and prepared and administered in vivo, under conditions such that production of the xcex1-2,8-sialyltransferase is suppressed. The base sequence of the synthetic oligonucleotide can be one that is in agreement with a part of the base sequence of the antisense strand of the DNA of the present invention or one that is modified without causing loss of its ability to inhibit the activity expression of the xcex1-2,8-sialyltransferase. When the triple helix technique is employed, the base sequence of the synthetic oligonucleotide can be designed based on the base sequence of both the sense and antisense strands.
It is also possible to detect the production of the xcex1-2,8-sialyltransferase of the present invention using the hybridization or PCR technique. For detecting the production of the xcex1-2,8-sialyltransferase of the present invention using the Northern hybridization or PCR technique, the DNA coding for the xcex1-2,8-sialyltransferase of the present invention or a synthetic oligonucleotide synthesized based on the base sequence thereof can be used. Northern hybridization and PCR can be carried out in a conventional manner [Sambrook, Fritsch and Maniatis: Molecular Cloningxe2x80x94A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989; Innis et al.: PCR protocols, Academic Press, 1990].